Large-scale purification and characterization of the Escherichia coli rep gene product

Lohman, Timothy M.; Chao, Kinlin; Green, J M; Sage, Stewart O.; Runyon, Gregory T.

doi:10.1016/s0021-9258(18)81778-1

Cited by 104 publications

(40 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Finally, the pooled fractions from this column were applied to a ssDNA cellulose column. Results show that only Rep was eluted between 0.8 and 1.1 M NaCl, consistent with the previous work . However, and following extensive washing with 2 M NaCl as described in the Materials and Methods, his-Rep and SSB were coeluted.…”

Section: Resultssupporting

confidence: 91%

“…Finally, as the kinetic parameters we obtain with the his-Rep and his-UvrD are within the same experimental error as those published previously, − we conclude that the tag has no discernible effect on protein activity.…”

Section: Resultssupporting

confidence: 89%

“…Analysis of the crystal structures reveals that the positioning of these tags should not impair enzyme function as the enzyme N-termini (thus the tags) are far from both the DNA and ATP-binding pockets (Supporting Information, Figure 1 and refs , ). The data presented below demonstrate that the activity of his enzymes is within the experimental error, the same as that of the untagged proteins. − …”

Section: Resultsmentioning

confidence: 69%

See 2 more Smart Citations

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

et al. 2019

View full text Add to dashboard Cite

The Rep and UvrD DNA helicases are proposed to act at stalled DNA replication forks to facilitate replication restart when RNA polymerase stalls forks. To clarify the role of these DNA helicases in fork rescue, we used a coupled spectrophotometric ATPase assay to determine how they act on model fork substrates. For both enzymes, activity is low on regressed fork structures, suggesting that they act prior to the regression step that generates a Holliday junction. In fact, the preferred cofactors for both enzymes are forks with a gap in the nascent leading strand, consistent with the 3′–5′ direction of translocation. Surprisingly, for Rep, this specificity is altered in the presence of stoichiometric amounts of a single-strand DNA-binding protein (SSB) relative to a fork with a gap in the nascent lagging strand. Even though Rep and UvrD are similar in structure, elevated concentrations of SSB inhibit Rep, but they have little to no effect on UvrD. Furthermore, Rep and UvrD antagonize one another at a fork. This is surprising given that these helicases have been shown to form a heterodimer and are proposed to act together to rescue an RNA polymerase-stalled fork. Consequently, the results herein indicate that although Rep and UvrD can act on similar fork substrates, they cannot function on the same fork simultaneously.

show abstract

Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 89%

Section: Resultsmentioning

confidence: 69%

See 1 more Smart Citation

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Potassium glutamate (KGlu), the primary cytoplasmic salt in Escherichia coli , accumulates at high concentrations (>0.3 M Glu – ) in response to osmotic stress. , In vitro , replacing Cl – with Glu – stabilizes folded proteins and greatly stabilizes protein–nucleic acid complexes. − While an increase in the concentration of KGlu destabilizes protein–nucleic acid complexes because of the dominance of the Coulombic (polyelectrolyte) effect of K + , binding constants of lac repressor–lac operator, IHF–H′ DNA, and closed RNA polymerase−λP R complexes all increase 30–40-fold when Cl – is replaced with Glu – − at a constant K + concentration, and the folding and assembly of a jaw/clamp to stabilize the RNAP–promoter open complex is favored by up to 100-fold …”

mentioning

confidence: 99%

Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9

Sengupta¹,

Pantel²,

Cheng³

et al. 2016

Biochemistry

View full text Add to dashboard Cite

In vitro, replacing KCl by potassium glutamate (KGlu), the E. coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu− in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to other stabilizers (KCl, KF, K2SO4) and destabilizers (GuHCl, GuHSCN). GuHSCN titrations at 20 °C, performed as a function of concentration of KGlu or other salt and monitored by NTL9-fluorescence, are analyzed to obtain r-values quantifying the Hofmeister salt concentration (m3)-dependence of the unfolding equilibrium constant Kobs (r-value = −dlnKobs/dm3 = (1/RT) dΔG°obs/dm3 = m-value/RT). r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model-compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 °C, we determine a KGlu r-value of 3.22 m−1, and K2SO4, KF, KCl, GuHCl and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38 and −3.00 m−1 respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values, and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 °C.

show abstract

“…We also demonstrated that the superhelicase-based VFA works at a wide range of salt and ATP concentrations (Figure S2). 22 Optimized conditions for helicase loading and unwinding were used for the rest of the study (Methods).…”

mentioning

confidence: 99%

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase

Hua¹,

Panja

Li³

et al. 2018

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Vectorial folding of RNA during transcription can produce intermediates with distinct biochemical activities. Here, we design an artificial minimal system to mimic cotranscriptional RNA folding in vitro. In this system, a presynthesized RNA molecule begins to fold from its 5'-end, as it is released from a heteroduplex by an engineered helicase that translocates on the complementary DNA strand in the 3'-to-5' direction. This chemically stabilized "superhelicase" Rep-X processively unwinds thousands of base pairs of DNA. The presynthesized RNA enables us to flexibly position fluorescent labels on the RNA for single-molecule fluorescence resonance energy transfer analysis and allows us to study real-time conformational dynamics during the vectorial folding process. We observed distinct signatures of the maiden secondary and tertiary folding of the Oryza sativa twister ribozyme. The maiden vectorial tertiary folding transitions occurred faster than Mg-induced refolding, but were also more prone to misfolding, likely due to sequential formation of alternative secondary structures. This novel assay can be applied to studying other kinetically controlled processes, such as riboswitch control and RNA-protein assembly.

show abstract

Large-scale purification and characterization of the Escherichia coli rep gene product

Cited by 104 publications

References 46 publications

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

Rep and UvrD Antagonize One Another at Stalled Replication Forks and This Is Exacerbated by SSB

Positioning the Intracellular Salt Potassium Glutamate in the Hofmeister Series by Chemical Unfolding Studies of NTL9

Mimicking Co-Transcriptional RNA Folding Using a Superhelicase

Contact Info

Product

Resources

About